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Parasitic infections are a widespread health problem and research of novel anthelmintic compounds is of the utmost importance. In this study we performed a virtual screening campaign by coupling ligand-based pharmacophore, homology modeling and molecular docking. The virtual screening campaign was conducted using a joined pool of the Drugbank database and a library of purchasable compounds in order to identify drug like compounds with similar pharmacological activity. Our aim was to identify compounds with a potential antihelmintic modulatory effect on nicotinic acetylcholine receptors (nAChR). We derived a 3D pharmacophore model based on the chemical features of known Ascaris suum nAChR modulators. To evaluate the in silico predictions, we tested selected hit compounds in contraction assays using somatic muscle flaps of the Ascaris suum neuromuscular tissue. We tested the modulatory effects of GSK575594A, diazepam and flumazenil hit compounds on nematode contractions induced by acetyl choline (ACh). The compound GSK575594A (3 μM) increased the E max by 21 % with the EC 50 dose ratio of 0.96. Diazepam (100 μM) decreased the E max by 15 % (1.11 g to 0.95 g) with the EC 50 ratio of 1.42 (shifted to the left from 11.25 to 7.93). Flumazenil decreased the EC 50 value (from 11.22 μM to 4.88 μM) value showing dose ratio of 2.30, and increased the E max by 4 % (from 1.54 g to 1.59 g). The observed biological activity was rationalized by molecular docking calculations. Docking scores were calculated against several binding sites within the Ascaris suum homology model. We constructed the homology model using the ACR-16 subunit sequence. The compound GSK575594A showed strong affinity for the intersubunit allosteric binding site within the nAChR transmembrane domain. The binding modes of diazepam and flumazenil suggest that these compounds have a comparable affinity for orthosteric and allosteric nAChR binding sites. The selected hit compounds displayed potential for further optimization as lead compounds. Therefore, such compounds may be useful in neutralizing the growing resistance of parasites to drugs, either alone or in combination with existing conventional anthelmintics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Startect(®) is a novel anthelmintic combination of derquantel and abamectin. It is hypothesized that derquantel and abamectin interact pharmacologically. We investigated the effects of derquantel, abamectin and their combination on somatic muscle nicotinic acetylcholine receptors and pharyngeal muscle glutamate gated chloride receptor channels of Ascaris suum. We used muscle-strips to test the effects of abamectin, derquantel, and abamectin+derquantel together on the contraction responses to different concentrations of acetylcholine. We found that abamectin reduced the response to acetylcholine, as did derquantel. In combination (abamectin+derquantel), inhibition of the higher acetylcholine concentration response was greater than the predicted additive effect. A two-micropipette current-clamp technique was used to study electrophysiological effects of the anthelmintics on: (1) acetylcholine responses in somatic muscle and; (2) on l-glutamate responses in pharyngeal preparations. On somatic muscle, derquantel (0.1-30μM) produced a potent (IC50 0.22, CI 0.18-0.28μM) reversible antagonism of acetylcholine depolarizations. Abamectin (0.3μM) produced a slow onset inhibition of acetylcholine depolarizations. We compared effects of abamectin and derquantel on muscle preparations pretreated for 30min with these drugs. The effect of the combination was significantly greater than the predicted additive effect of both drugs at higher acetylcholine concentrations. On the pharynx, application of derquantel produced no significant effect by itself or on responses to abamectin and l-glutamate. Abamectin increased the input conductance of the pharynx (EC50 0.42, CI 0.13-1.36μM). Our study demonstrates that abamectin and derquantel interact at nicotinic acetylcholine receptors on the somatic muscle and suggested synergism can occur., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Resistance of parasitic nematodes to the cholinergic anthelmintic levamisole is associated with a reduction in the proportion of time that acetylcholine receptor ion-channels are in the open state decreasing the response of nematode parasites to the drug. Here we examine electrophysiological and contractile responses to acetylcholine and the cholinergic agonist, levamisole, in Ascaris suum muscle looking for a pharmacological approach that may be developed to increase the response to cholinergic agonists. We found that short application of the FMRFamide, AF2, produced modulation (long lasting potentiation) of the peak membrane potential response to acetylcholine but not to levamisole. Since levamisole preferentially activates L-type acetylcholine receptors, we also tested the effect of nicotine (selective activator of N-type acetylcholine receptors) and bephenium (selective activator of B-type acetylcholine receptors) and found again no effect of AF2 on peak membrane potential responses. We then tested atropine on the AF2 potentiation of acetylcholine and found it to inhibit the peak potentiation suggesting that AF2 receptors interact with muscarinic receptors to produce the potentiation of acetylcholine. We saw similar atropine sensitive potentiation of acetylcholine responses in our muscle contraction experiments. The potentiation of the acetylcholine responses shows that nematode acetylcholine receptors are capable of a level of plasticity. A model involving calcium release from the sarcoplasmic reticulum, CaM Kinase, calcineurin, muscarinic receptors and AF2 receptors is proposed to explain our observations. These observations are important because they point to a pharmacological approach that may be developed to counter resistance to cholinergic anthelmintics.

The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum. However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle. Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition. As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition. However, although the free A. suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate. More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition. These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A. suum PDC to NADH inhibition during anaerobiosis.

Injection of Ascaris FMRFamide-like (AF) peptides and peptides encoded by genes in Caenorhabditis elegans were analyzed for effects on locomotion, body waveforms, and cAMP concentrations in adult female Ascaris suum. Injection of AF1 (KNEFIRFamide) or AF2 (KHEYLRFamide) inhibited the propagation of locomotory waves and reduced the number of waveforms, decreased the body length, and caused a large, long-lasting increase in cAMP. Muscle tissue was identified as a major source of the cAMP response induced by AF1. The AF1 analog AF1R6A did not affect cAMP levels by itself, but inhibited the cAMP response produced by AF1. AF8 (KSAYMRFamide) produced ventral coiling in the behavioral assay, and AF10 (GFGDEMSMPGVLRFamide) decreased the body length and increased the number of body waveforms. In dorsal muscle strips, AF10 produced a long-lasting contraction. Neither AF8 nor AF10 changed cAMP concentrations. AF17 (FDRDFMHFamide) increased body length and decreased cAMP. The neuropeptides encoded by C. elegans genes flp-4, flp-7, flp-9, and flp-13 produced paralysis and loss of waveforms, increased body length and, like AF17, decreased cAMP. Three new predicted peptides from C. elegans genome sequences were synthesized and tested. One produced ventral coiling but no change in cAMP; the other two gave no detectable responses. The fact that C. elegans neuropeptides produce behavioral and physiological effects in A. suum suggests that structurally related peptides may exist in A. suum. The profound changes in cAMP produced by some neuropeptides has important implications for understanding cAMP signaling and shows that neuropeptide-mediated signal transduction pathways are potential targets for anthelmintic drug development.

AF3 (AVPGVLRFamide) is an endogenous RFamide-like peptide isolated from the parasitic nematode Ascaris suum. It has a potent and long lasting excitatory effect in A. suum and Ascaridia galli. This is mediated by a mechanism independent of the nicotinic-like acetylcholine (ACh) receptor, which mediates excitatory transmission at the neuromuscular junction of both nematodes. In addition, AF3 has been found to sensitise A. suum muscle to the contractile effect of ACh. In this study, the involvement of the second messenger cAMP in mediating the action of AF3 on the somatic musculature of A. suum and A. galli has been investigated. Two approaches have been used; the effects of drugs which raise intracellular cAMP levels on the contractile responses to AF3 have been examined and biochemical assays have been used to measure the effects of AF3 on cAMP levels. AF3 contractions were inhibited in A. suum by 10 microM forskolin (by 22% of control; P < 0.05; n = 9) and by 500 microM isobutylmethylxanthine (IBMX, by 27% of control; P < 0.001; n = 6). AF3 decreased cAMP concentrations in A. suum somatic muscle (basal, 1721 +/- 134 pmol mg-1 protein; with 1 microM AF3, 1148 +/- 133 pmol mg-1 protein; P < 0.05, n = 5). AF3 (1 microM) also reduced the 10 microM forskolin induced potentiation of cAMP concentrations in A. suum (forskolin 3242 +/- 471 pmol mg-1 protein; forskolin and AF3, 1524 +/- 143 pmol mg-1 protein; P < 0.001, n = 6) and A. galli (forskolin 291 +/- 32 pmol mg-1 protein, forskolin +AF3, 185 +/- 12 pmol mg-1 protein; P < 0.005, n = 5). These data suggest that in both nematodes the contractile effect of AF3 is, at least in part, regulated by cAMP.